Predictive markers in elderly patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors: an array-based pharmacogenetic study.

So far, no reliable predictive clinicopathological markers of response to aromatase inhibitors (AIs) have been identified, and little is known regarding the role played by host genetics. To identify constitutive predictive markers, an array-based association study was performed in a cohort of 55 elderly hormone-dependent breast cancer (BC) patients treated with third-generation AIs. The array used in this study interrogates variants in 225 drug metabolism and disposition genes with documented functional significance. Six variants emerged as associated with response to AIs: three located in ABCG1, UGT2A1, SLCO3A1 with a good response, two in SLCO3A1 and one in ABCC4 with a poor response. Variants in the AI target CYP19A1 resulted associated with a favourable response only as haplotype; haplotypes with increased response association were also detected for ABCG1 and SLCO3A1. These results highlight the relevance of host genetics in the response to AIs and represent a first step toward precision medicine for elderly BC patients.

BRCA mutations and outcome in epithelial ovarian cancer (EOC): experience in ethnically diverse groups.

BACKGROUND: Epithelial ovarian cancer (EOC) patients with BRCA mutations have better prognosis than nonhereditary cases matched for histology and stage and age at diagnosis, especially Ashkenazi Jews (AJ). MATERIALS AND METHODS: We retrospectively reviewed data on 700 highly ethnically heterogeneous patients diagnosed with stage Ic-IV EOC and evaluated for BRCA status between 1995 and 2009 in American, Israeli, and Italian medical centers. RESULTS: The ethnicities of the 190 patients (median age 55.5 years, range 31-83 years) were AJ, Jewish non-Ashkenazi, Caucasian, African-American, Hispanic, or unknown. Ninety were BRCA1/2 carriers (71 BRCA1 and 19BRCA2). The most common mutations in AJ and non-AJ origins were 185delAG and 6174delT. Non-Jewish Caucasians exhibited the widest variation (>20 mutation subtypes). BRCA carriers had significantly prolonged median overall survival (93.6 months) compared with noncarriers (66.6 months; 95% confidence interval 44.5-91.7, P = 0.0081). There was no difference in progression-free survival. CONCLUSIONS: Our data demonstrate a wide variety of BRCA mutations in a highly ethnically diverse EOC population, and confirm that EOC BRCA mutation carriers have better prognosis with longer median survival than patients with nonhereditary disease. The contribution of unclassified BRCA variants to cancer etiology remains undetermined.

Targeted approaches to triple-negative breast cancer: current practice and future directions.

Triple-negative breast cancer (TNBC), that is breast cancer which stains negatively at immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2), comprises a particularly aggressive subtype of breast cancer, with high rate of early local and distant relapse. TNBC have demonstrated sensitivity to cytotoxic treatment regimens, but in the absence of HER2, ER and PR there is no benefit from hormonal therapy or trastuzumab. The lack of known specific molecular targets has promoted abundant research in order to find possible "vulnerabilities" in TNBC and the evaluation of novel biomarkers overcoming the traditional approach based on hormonal receptors and HER2-targeted therapy is one of the priorities in breast cancer research. Drugs under investigation can be broadly subdivided into four groups: (1) Agents that create DNA damage (i.e. cisplatin, cyclophosphamide); (2) Agents that inhibit poly (ADP-ribose) polymerase (PARP); (3) Tyrosin-kinase inhibitors and monoclonal antibodies; (4) Agents that inhibit downstream signals. Several preclinical and early phase clinical trials for the treatment or management of patients with triple-negative breast tumors are underway. Nonetheless, so far the major issue to deal with when trying to provide evidence for TNBC is the small numbers of the sample in the clinical studies and the retrospective nature of most of them. Future large studies could help in defining optimal treatment strategies for TNBC, both in the advanced setting as well as in the (neo) adjuvant setting.

Overall survival in BRCA-associated ovarian cancer: case-control study of an Italian series.

About 10% of all ovarian cancers are due to BRCA 1 and/or BRCA 2 mutations. Some studies have shown that patients belonging to this group have a better survival compared to sporadic groups but data are still inconclusive. The aim of this study was to investigate overall survival in patients with ovarian cancer and germ-line mutations in the BRCA1/2 genes in comparison to high-risk patients, defined as patients with ovarian cancer and a strong family history of breast and ovarian cancer, but who tested negative for the BRCA mutation. We collected all the clinical features and did follow-up. The two groups showed similar characteristics concerning age at diagnosis, histological type and stage. Grade 3 was more frequent in the BRCA group. Survival data did not show any advantage for the BRCA mutated group.

Rare presentation of endometrial carcinoma with singular bone metastasis.

The major symptom at diagnosis of endometrial cancer is post-menopausal bleeding; it is present in around 90% of cases. Singular bone metastasis is described as an uncommon site for endometrial cancer at diagnosis, showing in just 5-6% of cases. In this report we describe a rare presentation of a singular bone metastasis because of endometrial cancer of a woman with previous diagnosis of early breast cancer. A review of literature uncovered some cases of bone metastasis at presentation of endometrial cancer and that it can occur as first symptom of cancer before vaginal bleeding. This rare presentation of uterine cancer needs to be studied and described because it may be seen and needs a homogeneous treatment to improve survival.

In vivo activated T cells in rheumatoid synovitis. Analysis of Th1- and Th2-type cytokine production at clonal level in different stages of disease.

T-cell cytokines play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). Their detection in the joint, however, is impaired by the complex network present in the synovium. Although many synovial T cells show signs of previous activation, only a few express interleukin (IL)-2 receptor, marker of recent activation. The aim of this study was to analyse the cytokine production by in vivo activated (IL-2R +) T cells from RA at different stages of the disease. For this purpose, T cells were isolated from peripheral blood and synovial fluid of four patients with active RA, two at the onset of the disease, one in the early phase during treatment, one in long-lasting chronic phase. One patient was studied at the onset of the disease and 52 months later. Cells were initially expanded with a low dose of IL-2, cloned and analysed for cytokine production. The results showed a strong predominance of T helper (Th) 1 clones in the blood and a slight prevalence of Th0 clones in the joint of all the four patients. Interferon-gamma and IL-2 production was higher in the long-lasting RA, whereas IL-4 synthesis was prevalent in early RA. Enrichment in IL-10-producing clones was present only in the joint of the untreated patients. The longitudinal study confirmed the differences in cytokine production between early and late phases of disease. These data confirm that RA is mainly a Th1-driven condition. However, in vivo activated synovial T cells produce also Th2-type anti-inflammatory cytokines, such as IL-4 and IL-10. The synthesis of both cytokines is a feature of the very early phase of RA, although the selective recruitment of IL-10-producing T cells is quickly lost.

A 1.1-kb duplication in the p67-phox gene causes chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency that is caused by a functional defect of the NADPH oxidase of phagocytes, and that leads to severe recurrent infections. CGD results from the absence or the dysfunction of various components of NADPH oxidase, and autosomal recessive CGD with the lack of p67-phox (A67 CGD) is the rarest form of the disease. Identifying familiar mutations in subjects with A67 CGD provides the most reliable method of detecting carriers and is the basis for prenatal diagnosis. In the present study, we report the detailed characterization of the first duplication in the p67-phox gene identified in a 30-year-old patient affected by systemic aspergillosis attributable to p67-phox deficiency. We show that this new mutation involving exons 9 and 10 is the result of a tandem duplication of approximately 1.1 kb, which resulted from the juxtaposition of intron 8 to intron 10. We have sequenced both the junction fragment of this duplication and the corresponding wild-type regions and have found that the breakpoint regions in intron 8 and in intron 10 show limited homology. Our result suggests that this interchange arose as an illegitimate recombination event. As in other non-homologous rearrangements previously reported, the duplication breakpoints are located within the sequence motif 5'-CCAG-3' and its complement 5'-CTGG-3'.

Expression of plasminogen activator inhibitor-1 in human adipose tissue: a role for TNF-alpha?

Elevated plasminogen activator inhibitor-1 (PAI-1) plasma levels, responsible for reduced fibrinolysis, are associated with animal and human obesity and with increased cardiovascular disease. The expression of PAI-1 has been found recently in animal and human adipose tissue. Factors and mechanisms regulating such an expression remain to be elucidated. In omental and/or subcutaneous biopsies from obese non-diabetic patients, incubated in Medium 199, we have confirmed that human adipose tissue expresses PAI-1 protein and mRNA; furthermore we have demonstrated that such an expression is clearly evident also in collagenase isolated human adipocytes and that it is stimulated by incubation itself and enhanced by exogenous human tumor necrosis factor-alpha (h-TNF-alpha). Since human adipose tissue produces TNF-alpha, to further characterize the relationship of PAI-1 to TNF-alpha, human fat biopsies were also incubated with Pentoxifylline (PTX) or Genistein, both known to inhibit endogenous TNF-alpha through different mechanisms. PTX caused a dose-dependent decrease of basal PAI-1 protein release, reaching 80% maximal inhibitory effect at 10(-3)M, the same inhibitory effect caused by Genistein at 100 microg/ml. This was associated to a marked inhibition of PAI-1 mRNA and of endogenous TNF-alpha production. Furthermore, when human fat biopsies were incubated in the presence of polyclonal rabbit neutralizing anti-human TNF-alpha antibody (at a concentration able to inhibit 100 UI/ml human TNF-alpha activity), a modest but significant decrease of the incubation induced expression of PAI-1 mRNA was observed (19.8+/-19.0% decrease, P = 0.04, n = 7). In conclusion, the results of this study demonstrate that PAI-I expression is present in human isolated adipocytes and that it is enhanced in human adipose tissue in vitro by exogenous TNF-alpha. Furthermore our data support the possibility of a main role of endogenous TNF-alpha on human adipose tissue PAI-1 expression. This cytokine, produced by human adipose tissue and causing insulin resistance, may be a link in the clinical relationship between insulin-resistance syndrome and increased PAI-1 plasma levels.

GABA (gamma-amino-butyric acid) neurotransmission: identification and fine mapping of the human GABAB receptor gene.

GABA (gamma-amino-butyric acid) receptors are a family of proteins involved in the GABAergic neurotransmission of the mammalian central nervous system (CNS). They have physiological importance and clinical relevance in several diseases. We report the identification, cloning, and fine mapping of the human cDNA for GABAB receptor. A 4.2-Kb cDNA containing an open reading frame for a predicted protein of 960 aa was isolated from a fetal brain cDNA library. It had a strong identity (91.5%) with the rat GABAB receptor (rGB1A) nucleotide sequence, that corresponded to 98.6% identity at the amino acid level. Expression of the GABAB at the transcription level was detected by Northern analysis in all brain areas examined. The GABAB receptor has been mapped to human chromosome 6p21.3 within the HLA class I region close to the HLA-F gene. Susceptibility loci for multiple sclerosis, epilepsy, and schizophrenia have been suggested to map in this region.

Chronic parvovirus B19 infection induces the production of anti-virus antibodies with autoantigen binding properties.

Human parvovirus B19 infection in adults shows some clinical features similar to those found in autoimmune connective tissue diseases. To better clarify the relationship between viral infection and autoimmunity, we have evaluated the ability of anti-parvovirus antibodies to specifically recognize autoantigens in ten patients with chronic symmetric arthritis resembling rheumatoid arthritis or with recurrent episodes of arthritis and cutaneous manifestations and persistence of specific IgM antibodies against B19 parvovirus. We synthetized a 24-amino acid immunodominant peptide corresponding to a part of the virus protein 1 and virus protein 2 overlapping region. The peptide has been used to test patients' sera at different time points with an enzyme-linked immunosorbent assay (ELISA) and to purify anti-virus antibodies by affinity chromatography on a peptide-Sepharose column. Eluted immunoglobulins recognized the B19 peptide in both direct and competitive ELISA. Affinity-purified anti-parvovirus antibodies were then tested on a panel of autoantigens including human keratin, collagen type II, thyreoglobulin, single-strand (ss)DNA, cardiolipin and ribonucleoprotein antigen Sm. Eluted antibodies specifically recognized keratin, collagen type II, ssDNA and cardiolipin. Autoantibody activity was not detected in the immunoglobulin fraction after complete removal of anti-peptide antibodies and in antibodies eluted from normal donors. Epstein-Barr virus-transformed cell clones obtained from two subjects produced antibodies which simultaneously recognize the viral peptide and several autoantigens. To further confirm the role of the virus in inducing an autoantibody response, eight BALB/c mice were immunized with the viral peptide coupled to a carrier protein. Autoantibody activity against keratin, collagen II, cardiolipin and ssDNA was detected in six of the eight mice which developed a strong anti-virus response. Together, these data indicate that B19 parvovirus may be linked to the induction of an autoimmune response.

Analysis of the T cell receptor repertoire in rheumatoid arthritis.

OBJECTIVE: To evaluate whether there is a restricted T cell receptor repertoire in rheumatoid synovium and to analyse the CDR3 region of the V beta families found to be more expressed in the synovial membrane than in the peripheral blood, in order to ascertain the presence of clonotypic expansion of T lymphocytes. METHODS: The level of expression of individual V beta and V alpha families of the TCR was evaluated in paired synovial membrane and peripheral blood T cells from 8 female patients affected by rheumatoid arthritis, using the RT-PCR method. Nucleotide sequences of the CDR3 region of some V beta families were analysed in order to identify the presence of conserved sequences. Sequencing was carried out with the dideoxy chain termination method using modified T7 DNA polymerase. RESULTS: All of the V alpha and V beta families were amplified in both compartments of the 8 patients. Four patients did not show any preferential expression of the TCR alpha or beta chains in synovium compared with peripheral blood. The other 4 patients showed increased expression of one or more V alpha and/or V beta families in the synovium. We did not find any correlation between the duration of disease, rheumatoid factor status, HLA-DR type and the V gene families which were elevated in the synovium. Analysis of the CDR3 region showed the presence of conserved amino acid sequences in the synovium, but not in the peripheral blood. CONCLUSION: The V families found to be increased in 4 of the 8 patients studied were different, except for V beta 1 which was more highly expressed in 2 patients. The presence of conserved amino acid sequences in the CDR3 region is consistent with an antigen-driven T cell expansion at the site of autoimmune inflammation. These findings do not support our original hypothesis of the possible usefulness of therapy based on the inactivation or elimination of presumed pathogenic T cells using TCR-derived peptides or monoclonal antibodies against particular TCRs.

Linkage analysis of 6p21 polymorphic markers and the hereditary hemochromatosis: localization of the gene centromeric to HLA-F.

Hereditary Hemochromatosis (HFE) is one of the most common inherited disorders with an estimated frequency of homozygous patients of 0.002-0.0045. The disease is characterized by increased intestinal iron absorption and progressive iron overload. Affected subjects show clinical symptoms of parenchymal organ damage after the third-fourth decade of life and have a 200 fold increased risk of developing hepatocellular carcinoma. Early diagnosis and treatment prevent complications and may normalize life expectancy of patients. The biochemical and genetic defects leading to progressive iron accumulation are still unknown, but the HFE gene is tightly linked to HLA complex on the short arm of chromosome 6. Utilizing HLA serotypes and the study of several polymorphic markers of 6p21, a linkage analysis of the disease locus was performed in a series of Italian hemochromatosis families. The data obtained by linkage analysis and the study of a family with a double recombinant allowed us to better define the HFE gene location with respect to HLA-class I A and F loci.

Evidence of c-myc gene abnormalities in mediastinal large B-cell lymphoma of young adult age.

Six cases of mediastinal large B-cell lymphoma (MLCL) with sclerosis were analyzed for the presence and patterns of c-myc and bcl-2 loci rearrangements, and for the presence of Epstein-Barr virus DNA sequences by Southern blot hybridization, c-myc gene alterations were found in three of six cases. Two cases showed the presence of mutations or small rearrangements at the 3' end of the first exon. The c-myc gene abnormalities found in these two cases are similar to those observed in the translocation 8;14 of the endemic Burkitt's lymphomas or in its variants t(2;8) and t(8;22). A third case showed a major rearrangement of c-myc gene, with truncation within its first intron, similar to those observed in sporadic Burkitt's and in acquired immunodeficiency-associated lymphomas. None of the cases displayed bcl-2 gene rearrangements or contained viral sequences. Our data suggest a possible role for a translocation-mediated c-myc activation in the pathogenesis of MLCL. Conversely, bcl-2 gene and Epstein-Barr virus do not appear to be involved in the pathogenesis of these peculiar lymphomas. The association between c-myc structural modifications and MLCL also seems to be of relevance in light of the peculiar tendency of this tumor to involve unusual extranodal site (eg, kidney), reminiscent of the spreading attitude of Burkitt's limphomas.